BACKGROUND: Whereas multi-species biofilm reactors are commonly used for the treatment of liquid and solid wastes, new strategies are progressing for the development of single species biofilm for the ... [more ▼]

BACKGROUND: Whereas multi-species biofilm reactors are commonly used for the treatment of liquid and solid wastes, new strategies are progressing for the development of single species biofilm for the production of high-value metabolites. Technically, this new concept relies on the design of bioreactors able to promote biofilm formation and on the identification of the key physico-chemical parameters involved in biofilm formation. RESULTS: An experimental setting comprising a liquid continuously recirculated on a metal structured packing has been used to promote Bacillus subtilis GA1 biofilm formation. The colonization of the packing has been visualized non-invasively by X-ray tomography. This analysis revealed an uneven, conical, distribution of the biofilm inside the packing. Compared with a submerged culture carried out in a stirred tank reactor, significant modification of the lipopeptide profile has been observed in the biofilm reactorwith the disappearance of fengycin and iturin fractions and an increase of the surfactin fraction. In addition, considering the biofilm reactor design, no foam formation has been observed during the culture. CONCLUSIONS: The configuration of this biofilm reactor set-up allows for a higher surfactin production by comparison with a submerged culture while avoiding foam formation. Additionally, scale-up could easily be performed by increasing the number of packing elements. [less ▲]

This report is to our knowledge the first to study plant growth promotion and biocontrol characteristics of Bacillus isolates from extreme environments of Eastern Algeria. Seven isolates of 14 (50 %) were ... [more ▼]

This report is to our knowledge the first to study plant growth promotion and biocontrol characteristics of Bacillus isolates from extreme environments of Eastern Algeria. Seven isolates of 14 (50 %) were screened for their ability to inhibit growth of some phytopathogenic fungi on PDA and some roots exudates. The bacteria identification based on 16S r-RNA and gyrase-A gene sequence analysis showed that 71 % of the screened isolates belonged to Bacillus amyloliquefaciens and the rest were closely related to B. atrophaeus and B. mojavensis. Most of them had high spore yields (22 x 108-27 x 108 spores/ml). They produced protease and cellulase cell wall-degrading enzymes while the chitinase activity was only observed in the B. atrophaeus (6SEL). A wide variety of lipopeptides homologous was detected by liquid chromatography-electrospray ionization-mass spectrometry analysis. Interestingly, some additional peaks with new masses were characterized, which may correspond to new fengycin classes. The isolates produced siderophores and indole-3- acetic acid phytohormone. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. atrophaeus (6SEL) significantly increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05). These results suggest that these isolates may be used further as bio-inoculants to improve crop systems. [less ▲]

Generally, an antibiotic is thought to have a role in antagonism simply because the producing strain is known to exhibit a potential for pathogen growth inhibition. Some genetic approaches such as PCR ... [more ▼]

Generally, an antibiotic is thought to have a role in antagonism simply because the producing strain is known to exhibit a potential for pathogen growth inhibition. Some genetic approaches such as PCR using specific primers or genome mining using known sequence data of close relatives are also used. Nevertheless, none of these methods allows stating for a link between a specific compound and the observed antagonism. Yet MALDI Mass Spectrometry Imaging (MSI) is a powerful tool to decipher the chemical messengers exchanged by two protagonists [1,2,3;]. Tandem mass spectrometry (MS/MS) may be also used, either on extracts [2,3] or directly on the microbial colonies [4]. The presentation will thus be focused on two examples of application of MALDI MSI combined to in situ tandem mass spectrometry. The first presented case will be the antagonism between soilborne strain Paenibacillus polymyxa Pp56 and the fungal phytopathogen Fusarium oxysporum. Using MALDI MSI, we were able to precisely localize each detected antibiotic, allowing discriminating which LI-F lipopeptides (fusaricidin) were really active against the pathogen progression. Besides, the use of in situ MS/MS allowed us to sequence the peptide moiety of several LI-F lipopeptides, showing that some of them are actually a mixture of several forms. The second example concerns the metabolites that are released by Bacillus amyloliquefaciens S499 cells following their inoculation on 7 days old tomato roots. We developed specific bioassays for time-course monitoring by MALDI MSI. First analyses revealed an efficient secretion of surfactin by Bacillus cells after 3 days when colonization as biofilm-structured populations is well established. Even if the composition of antibiotic mixture does not greatly evolve over time, after long incubation periods (32 or 35 days post inoculation), new series of compounds are detected in the tomato root -surrounding medium. Structural analysis based on exact mass measurements and MS/MS experiments, performed directly on the semi-solid agar medium, allowed us to identify these compounds as new variants of surfactins. [1] Barger, S., et al., Anton Leeuw Int J G, 2012, 102, 435-445. [2] Hoefler, B. C., et al,. Natl Acad Sci USA, 2012, 109, 13082-13087. [3] Moree, W. J., et al., Natl Acad Sci USA, 2012, 109, 13811-13816. [4] Debois, D., et al., J Am Soc Mass Spectrom. 2013, 24, 1202-1213 [less ▲]

Biosurfactants which are surface active molecules produced by micro-organisms present a wide structural diversity (glycolipids, lipoaminoacids, lipopeptides, polymers,...) and numerous advantages compared to their chemically synthesized counterparts. Among glycolipids, rhamnolipids which are secondary metabolites produced mainly by strains of Pseudomonas aeruginosa, have drawn particular attention as they have several interesting biological properties such as antimicrobial, antiphytoviral, zoosporicidal and plant defense elicitor activities [1-3]. It is generally recognized that these activities must be linked to the interaction of these molecules with constituents of biological membranes [4] but the detailed mechanism is far from being fully understood. In our laboratory, new rhamnolipids with various chain lengths and with or without a terminal carboxylic acid function were obtained via the development of a synthesis procedure consisting of two biocatalyzed steps involving naringinase and lipase [5]. The objective of this work was to investigate their interaction with model membranes in relation with their structure in order to give insight about the mechanism of their biological action. A range of complementary experimental and modelling methods was used to analyze their interaction with membrane models. Results reveal differential interaction with lipids according to the structure of the rhamnolipid. The nature of the lipid is also a key parameter for the ınteractions. [less ▲]

Glycolipid surfactants constitute a class of natural compound having interest in biological field such as antifungal, antiviral and plant-elicitor and for pharmaceutical formulation as well as for food ... [more ▼]

Glycolipid surfactants constitute a class of natural compound having interest in biological field such as antifungal, antiviral and plant-elicitor and for pharmaceutical formulation as well as for food and cosmetic field. Their action may be modulated through their interactions with plasma membranes of target cells and more particularly by their interactions with membrane lipid molecules. Among glycolipid surfactants, bolaforms constitute an important class; they are composed of two hydrophilic heads connected by a hydrophobic carbon segment.Their interest lies mainly in the development of the efficient and low cost lipid-based drug delivery systems. In this context, our work was focused on two sugar-based bolaforms. They are composed by two identical hydrophilic head constituted by xylose ( BolaX) or rhamnose ( BolaR), connected by an ether link to a hydrocarbon segment with an insaturation. The interactions of  BolaX and BolaR with model phopsholipid and phospholipid/sterol model membranes (Langmuir monolayers at the air-water interface and multilamellar vesicles) were investigated with biophysical and in silico approaches. Our results indicate that both bolaforms interact with model membranes at the level of hydrocarbon chain and, at the phosphate and the carbonyl group of phospholipids. The presence of sterol in the system has an influence on insertion of bolaforms and change slightly the nature of the interactions. The insertion of BolaR within a phospholipid bilayer was deeper than that of  BolaX and its interactions with phospholipids were energetically more favorable, suggesting an important role of the nature of sugar residue. [less ▲]

The rhizobacterium Pseudomonas putida BTP1 stimulates induced systemic resistance (ISR) in tomato. A previous work showed that the resistance is associated in leaves with the induction of the first enzyme ... [more ▼]

The rhizobacterium Pseudomonas putida BTP1 stimulates induced systemic resistance (ISR) in tomato. A previous work showed that the resistance is associated in leaves with the induction of the first enzyme of the oxylipin pathway, the lipoxygenase (LOX), leading to a faster accumulation of its product, the free 13-hydroperoxy octadecatrienoic acid (13-HPOT), 2 days after Botrytis cinerea inoculation. In the present study, we further investigated the stimulation of the oxylipin pathway: metabolites and enzymes of the pathway were analyzed to understand the fate of the 13-HPOT in ISR. Actually the stimulation began upstream the LOX: free linolenic acid accumulated faster in P. putida BTP1-treated plants than in control. Downstream, the LOX products 13-fatty acid hydroperoxides esterified to galactolipids and phospholipids were more abundant in bacterized plants than in control before infection. These metabolites could constitute a pool that will be used after pathogen attack to produce free fungitoxic metabolites through the action of phospholipase A2, which is enhanced in bacterized plants upon infection. Enzymatic branches which can use as substrate the fatty acid hydroperoxides were differentially regulated in bacterized plants in comparison to control plants, so as to lead to the accumulation of the most fungitoxic compounds against B. cinerea. Our study, which is the first to demonstrate the accumulation of an esterified defense metabolite during rhizobacteria-mediated induced systemic resistance, showed that the oxylipin pathway is differentially regulated. It suggests that this allows the plant to prepare to a future infection, and to respond faster and in a more effective way to B. cinerea invasion. [less ▲]

in Journal of the American Society for Mass Spectrometry (2013), 24(8), 1202-1213

Nowadays, microorganisms are more and more often used as biocontrol agents for crop protection against diseases. Among them, bacteria of Bacillus and Paenibacillus genders are already used as commercial ... [more ▼]

Nowadays, microorganisms are more and more often used as biocontrol agents for crop protection against diseases. Among them, bacteria of Bacillus and Paenibacillus genders are already used as commercial biocontrol agents. Their mode of action is supposed to be related to their production of antibiotics, such as cyclic lipopeptides, which exhibit great antimicrobial activities. We chose to work with a Paenibacillus polymyxa strain (Pp56) very resistant to various microorganisms. The bacteria were grown simultaneously with Fusarium oxysporum and we applied matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry to identify the antibiotics compounds present in the fungus growth inhibition area. We, therefore, identified fusaricidins A, B, and C and numerous members of the LI-F antibiotics family. MALDIFTICR mass spectrometry imaging was then used to follow the diffusion of lipopeptides involved in the inhibitory activity over time. We analyzed the molecular content of the inhibitory area at different Pp56 and Fusarium incubation durations and concluded that some lipopeptides such as fusaricidin B and a mixture of LI-F05b/06b/08a were mainly involved in the defense mechanism of Pp56. Our study confirms that MALDI imaging may be a powerful tool to quickly determine which molecular species is involved in an antagonism with another microorganism, avoiding time-consuming steps of extraction, purification, and activity tests, which are still commonly used in microbiology. [less ▲]

Some non-pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in ... [more ▼]

Some non-pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in soil (1). To initiate both phenomena leading to biocontrol activity, microorganisms use plant exudates to grow on roots and to produce in-situ active compounds. In Bacilli, cyclic lipopeptides of the surfactin, iturin and fengycin families represent important antibiotics involved in biocontrol (2). Recent studies in microbiology allowed a better understanding of plant microorganism interactions but few has been done at the molecular level. In this study, MALDI MS imaging has been used to study the nature of the secreted lipopeptide molecules, their relative quantity and their distribution in the root’s environment.Disinfected tomato seeds were first germinated at 28°C in sterile conditions for germination. Seedlings were then placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution containing 1,75% of agar and treated with freshly-grown cells of Bacillus amyloliquefaciens S499. Petri dishes were incubated at 28°C with a 16h photoperiod. Different growth / incubation durations were studied: 10/3; 13/7; 21/14 and 39/32. For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. (HCCA, 5mg/mL in ACN/0.2% TFA 70:30) was used as matrix. UltraFlex II TOF/TOF and Solarix FT-ICR mass spectrometers were used to record molecular cartographies and perform MS/MS experiments for structural analysis purposes. The average mass spectra recorded around the tomato root (2-3 mm on both sides of the root) showed that lipopeptides were major compounds detected on the agar. The relative intensity of lipopeptides families varied with respect to the age of the root/biofilm system. In the 10/3 system, 3 homologues of surfactins were essentially detected (C13, C14 and C15), with very few iturins and fengycins. Their localizations were identical, whatever the considered homologue. Then the production of iturin and fengycin families increases in older systems (13/7 and 21/14) and a novel homologue of surfactin is detected (C12). Some variations in localizations within families may be observed (around the root or at the close vicinity of it in function of the considered homologue or alkali adduct). Then for the oldest system we studied, iturins and fengycins are not detected anymore and the localization of surfactins is less precise. In the 39/32 system, we also detected unknown compounds at 986.6, 1000.6, 1014.7 and 1028.7 m/z. The mass range of these compounds allied to the mass difference between two consecutive ion peaks let us think that these unknown compounds could be a new lipopeptide family. Tandem mass spectrometry experiments, performed on the dried culture medium, allowed to partially sequence these new lipopeptides. MS/MS results allied to exact mass measurements and isotopic pattern simulation give good confidence in the chemical structure we suggest. Nevertheless, to fully identify these new variants of surfactin, micro-extractions followed by (LC)-nano-ESI-MS/MS using a LESA module are in progress. MALDI Mass Spectrometry Imaging becomes a tool to decipher inter-species molecular communication. [less ▲]